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Related Concept Videos

G-protein Coupled Receptors01:21

G-protein Coupled Receptors

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G-protein coupled receptors are ligand binding receptors that indirectly affect changes in the cell. The actual receptor is a single polypeptide that transverses the cell membrane seven times creating intracellular and extracellular loops. The extracellular loops create a ligand specific pocket which binds to neurotransmitters or hormones. The intracellular loops holds onto the G-protein.
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G Protein-coupled Receptors01:15

G Protein-coupled Receptors

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G Protein-Coupled Receptors or GPCRs are membrane-bound receptors that transiently associate with heterotrimeric G proteins and induce an appropriate response to sensory stimuli such as light, odors, hormones, cytokines, or neurotransmitters.
GPCRs are also called heptahelical, 7TM, or serpentine receptors, and consist of seven (H1-H7) transmembrane alpha-helices that span the bilayer to form a cylindrical core. The transmembrane helices are connected by three extracellular loops and three...
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Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro
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Split-Tobacco Etch Virus (Split-TEV) Method in G Protein-Coupled Receptor Interacting Proteins.

Marta Alonso-Gardón1,2, Raúl Estévez3,4

  • 1Unitat de Fisiologia, Departament de Ciències Fisiològiques, Genes Disease and Therapy Program, IDIBELL-Institute of Neurosciences, Universitat de Barcelona, L'Hospitalet de Llobregat, Spain.

Methods in Molecular Biology (Clifton, N.J.)
|June 4, 2021
PubMed
Summary

This study presents a modified Split-TEV assay to accurately identify protein-protein interactions, specifically for G protein-coupled receptors (GPCRs) in mammalian cells. The enhanced method improves specificity, reducing false positives from membrane protein aggregation.

Keywords:
G protein-coupled receptorMembrane proteinsProteinSplit-TEVprotein interaction

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Background:

  • Protein-protein interactions are crucial for cellular functions.
  • The Split-Tobacco Etch Virus (TEV) protease assay identifies these interactions by reconstituting protease activity.
  • Unspecific interactions, particularly with membrane proteins like G protein-coupled receptors (GPCRs), can be a challenge.

Purpose of the Study:

  • To develop a more specific variation of the Split-TEV assay.
  • To improve the identification of protein-protein interactions involving GPCRs.
  • To provide a robust tool for drug discovery.

Main Methods:

  • A modified Split-TEV assay was developed.
  • The assay fuses TEV protease fragments to candidate proteins, including GPCRs.
  • Reconstitution of TEV protease activity upon interaction triggers reporter gene expression.

Main Results:

  • The modified Split-TEV assay demonstrates increased specificity for detecting protein-protein interactions.
  • The method effectively monitors interactions involving GPCRs.
  • The assay is robust and easy to implement.

Conclusions:

  • The enhanced Split-TEV assay offers improved specificity for studying GPCR interactions.
  • This method is a valuable tool for drug discovery and understanding cellular signaling pathways.
  • The assay overcomes limitations of previous methods regarding membrane protein interactions.